This invention relates to a novel substituted isoxazolylthiophene compound and a pharmaceutical composition comprising the same as an active ingredient, which is useful for enhancing the action of cell differentiation induction factors.
Compounds which have been reported as being therapeutically or prophylactically effective against bone diseases or nerve diseases by enhancing the action of the cell differentiation induction factors present in or administered to a living body include fused thiophene derivatives disclosed in W098/09958, but no such report covers the compounds of the present invention.
We extensively studied and found that certain substituted isoxazolylthiophene compounds are effective for treatment or prevention of bone diseases or nerve diseases, and finally completed the invention.
More specifically, the present invention is directed to a substituted isoxazolylthiophene compound represented by the formula (I) 
wherein R1 and R2 individually represent an alkyl group of 1-5 carbon atoms, R3 represents a cyano group or a group CONR5R6 (in which R5 and R6 individually represent a hydrogen atom or an alkyl group of 1-10 carbon atoms), R4 represents an alkyl group of 1-5 carbon atoms or a phenyl group, and n is an integer of 0-2, or a salt thereof.
The alkyl group of 1-5 carbon atoms used herein means a straight or branched alkyl group and includes, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group and an n-pentyl group.
The alkyl group of 1-10 carbon atoms as used herein means a straight or branched alkyl group and includes, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, an n-pentyl group, an n-hexyl group and an n-octyl group.
The salt as used herein may include salts with pharmaceutically acceptable acids (such as hydrochloric acid, sulfuric acid, nitric acid, tartaric acid, citric acid, maleic acid, fumaric acid, etc.), as well as their hydrates.
The invention may further encompass the compounds which are capable of producing the active compounds of this invention through in vivo metabolism after administration, or the compounds which are capable of producing the active compounds per se as produced by the metabolism of the compounds of the invention through in vivo metabolism.
The compounds (I) of the invention may be prepared, for example, according to the following processes:
1) The compounds (I) wherein R3 is a cyano group and n is 0 may be prepared, for example, according to the process as illustrated by Reaction Scheme 1. 
In the Reaction Scheme 1, R1 and R2 have the same meanings as defined above, and R7 is a group COCH3, COCH2R4 or CH2COR4 (wherein R4 has the same meaning as defined above).
The Reaction Scheme 1 will be explained in detail below.
1)-(1) The present compounds (Ia) and (Ib) may be prepared using as a starting material the diketone compound (II) wherein R7 represents COCH3. 
More specifically, the diketone compound (II) wherein R7 represents COCH3 may be condensed with carbon disulfide (CS2) in the presence of a base and then the resulting condensed product may be converted to the thiophene compound (III) wherein R7 represents COCH3 by thioetherification of one of the sulfur atoms derived from carbon disulfide with a haloacetonitrile such as chloroacetonitrile or bromoacetonitrile and the remaining sulfur atom with R2xe2x80x94X1 (wherein R2 has the same meaning as defined above and X1 is a leaving group such as a halogen atom, e.g., a chlorine atom or a bromine atom or a methylsulfoxy group), simultaneously with intramolecular cyclization reaction.
The base which may be used in this reaction may include alkali metal hydroxides (lithium hydroxide, sodium hydroxide, potassium hydroxide, etc.), alkali metal carbonates (lithium carbonate, sodium carbonate, potassium carbonate, etc.), alkali metal hydrogencarbonates (sodium hydrogencarbonate, potassium hydrogencarbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), inorganic bases (metallic sodium, metallic potassium, sodium amide, etc.), alkali metal alkoxides (sodium methoxide, sodium ethoxide, potassium t-butoxide, etc.), organic bases (triethylamine, diisopropylethylamine, tri-n-butylamine, 1,5-diazabicyclo[4.3.0]-5-nonene, 1,8-diazabicyclo[5.4.0]-7-undecene, pyridine, N,N -dimethylaminopyridine, etc.), organometallic compounds (n-butyl lithium, s-butyl lithium, t-butyl lithium, lithium diisopropylamide, sodium bis(trimethylsilyl)amide, etc.) and the like.
The reaction may be carried out in the presence or absence of a solvent. The solvent which may be used includes methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, dioxane, tetrahydrofuran, diethyl ether, petroleum ether, n-hexane, cyclohexane, benzene, toluene, xylene, chlorobenzene, pyridine, ethyl acetate, N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform, carbon tetrachloride, water and the like.
The base and solvent to be used, as well as use or no use of the solvent should be properly selected depending on the substrates and reaction parameters used in the reaction.
Then, R7 of the thiophene compound (III) wherein R7 is a group COCH3 is condensed with an activated carboxylic acid derivative represented by R4xe2x80x94COOH (wherein R4 has the same meaning as above) such as an alkyl ester, e.g., methyl ester or ethyl ester or an acid halide or an acid anhydride, to convert R7 to a group COCH2COR4. Subsequent condensed cyclization reaction using hydroxylamine or a derivative thereof may produce the compound (Ia) or (Ib) of this invention.
This condensation reaction is preferably carried out in the presence of a base. The base which may be used in this reaction includes alkali metal hydroxides (lithium hydroxide, sodium hydroxide, potassium hydroxide, etc.), alkali metal carbonates (lithium carbonate, sodium carbonate, potassium carbonate, etc.), alkali metal hydrogencarbonates (sodium hydrogencarbonate, potassium hydrogencarbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), inorganic bases (metallic sodium, metallic potassium, sodium amide, etc.), alkali metal alkoxides (sodium methoxide, sodium ethoxide, potassium t-butoxide, etc.), organic bases (triethylamine, diisopropylethylamine, tri-n-butylamine, 1,5-diazabicyclo[4.3.0]-5-nonene, 1,8-diazabicyclo[5.4.0]-7-undecene, pyridine, N,N-dimethylaminopyridine, etc.), organometallic compounds (n-butyl lithium, s-butyl lithium, lithium diisopropylamide, sodium bis(trimethylsilyl)amide, etc.) and the like.
The reaction may be carried out in the presence or absence of a solvent. The solvent which may be used includes methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, dioxane, tetrahydrofuran, diethyl ether, petroleum ether, n-hexane, cyclohexane, benzene, toluene, xylene, chlorobenzene, pyridine, ethyl acetate, N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform, carbon terachloride, water and the like.
The hydroxylamine which is used for condensed cyclization reaction may be in the form of a salt with hydrochloric acid, sulfuric acid or the like, and the reaction is preferably carried out in the presence of a base. The bases which may be used in this reaction include alkali metal hydroxides (lithium hydroxide, sodium hydroxide, potassium hydroxide, etc.), alkali metal carbonates (lithium carbonate, sodium carbonate, potassium carbonate, etc.), alkali metal hydrogencarbonates (sodium hydrogencarbonate, potassium hydrogencarbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), inorganic bases (metallic sodium, metallic potassium, sodium amide, etc.), alkali metal alkoxides (sodium methoxide, sodium ethoxide, potassium t-butoxide, etc.), organic bases (triethylamine, diisopropylethylamine, tri-n-butylamine, 1,5-diazabicyclo[4.3.0]-5-nonene, 1,8-diazabicyclo[5.4.0]-7-undecene, pyridine, N,N-dimethylaminopyridine, etc.), organometallic compounds (n-butyl lithium, s-butyl lithium, lithium diisopropylamide, sodium bis(trimethylsilyl)amide, etc.) and the like.
The reaction may be carried out in the presence or absence of a solvent. The solvent which may be used includes methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, dioxane, tetrahydrofuran, diethyl ether, petroleum ether, n-hexane, cyclohexane, benzene, toluene, xylene, chlorobenzene, pyridine, ethyl acetate, N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform, carbon terachloride, water and the like.
The reagent and solvent to be used, as well as use or no use of the solvent should be properly selected for each of these reactions, depending on the substrates and reaction parameters used.
1)-(2) The compounds (Ic-If) of the invention may be prepared according to the Reaction Scheme 1 using as a starting material the diketone compound (II) wherein R7 represents COCH2R4 or CH2COR4. 
More specifically, the thiophene compound (III) wherein R7 represents COCH2R4 or CH2COR4 may be prepared according to the process of 1)-(1) using the diketone compound (II) wherein R7 represents a group COCH2R4 or CH2COR4.
The compounds (Ic-If) of the invention may be prepared by converting R7 of the thiophene compound (III) wherein R7 represents a group COCH2R4 or CH2COR4 to formyl, halomethylene, alkoxymethylene or aminomethylene, followed by subsequent condensed cyclization reaction using hydroxylamine or a derivative thereof.
The formylation reaction may be carried out by a process wherein a formyl ester such as methyl formate or ethyl formate or carbon monooxide is condensed in the presence of a base, or a process wherein the carbonyl group of R7 is converted to an enamine with secondary amine such as dimethylamine, pyrrolidine or morpholine followed by the reaction with an N,N-dimethylformamide derivative such as N,N-dimethylformamide, N,N-dimethylformamide dimethyl acetal or t-butoxy(dimethylamino)methane in the presence of 10 phosgene, phosphorus oxychloride, oxalyl chloride or the like.
The aminomethylene-forming reaction may be carried out using an N,N-dimethylformamide derivative such as N,N-dimethylformamide, N,N-dimethylformamide dimethyl acetal or t-butoxy(dimethylamino)methane.
The halomethylene-forming reaction may be carried out by converting the carbonyl group of R7 to an enamine with a secondary amine such as dimethylamine, pyrrolidine or morpholine and subsequent reaction with a haloform such as chloroform in the presence of a base.
The alkoxymethylene-forming reaction may be carried out by heating in the presence of an alkyl orthoformate such as methyl orthoformate or ethyl orthoformate and acetic anhydride.
The aminomethylene-forming reaction may be carried out using an N,N-dimethylformamide derivative such as N,N-dimethylformamide dimethyl acetal or t-butoxy(dimethylamino)methane.
The bases which may be used in the formylation and the halomethylene-forming reaction include alkali metal hydroxides (lithium hydroxide, sodium hydroxide, potassium hydroxide, etc.), alkali metal carbonates (lithium carbonate, sodium carbonate, potassium carbonate, etc.), alkali metal hydrogencarbonates (sodium hydrogencarbonate, potassium hydrogencarbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), inorganic bases (metallic sodium, metallic potassium, etc.), alkali metal acetate (sodium acetate, etc.), alkali metal alkoxides (sodium methoxide, sodium ethoxide, potassium t-butoxide, etc.), organic bases (triethylamine, diisopropylethylamine, tri-n-butylamine, 1,5-diazabicyclo[4.3.0]-5-nonene, 1,8-diazabicyclo[5.4.0]-7-undecene, pyridine, N,N-dimethylaminopyridine, etc.), organometallic compounds (n-butyl lithium, s-butyl lithium, t-butyl lithium, lithium diisopropylamide, sodium bis(tri-methylsilyl)amide, etc.) and the like.
The formylation reaction, halomethylene-forming reaction, alkoxymethylene-forming reaction and aminomethylene-forming reaction may be carried out in the presence or absence of a solvent. The solvent which may be used includes methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, dioxane, tetrahydrofuran, diethyl ether, petroleum ether, n-hexane, cyclohexane, benzene, toluene, xylene, chlorobenzene, pyridine, ethyl acetate, N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform, carbon tetrachloride, water and the like.
The reagent and solvent to be used in each of the reactions, as well as use or no use of the solvent, should be properly selected, depending on the substrates and reaction parameters used.
2) The compound (Ig) of the invention, which is the compound (I) wherein R3 represents a group CONR5R6 (wherein R5 and R6 have the same meanings as defined above) and n is 0, may be prepared by hydrolysis of the cyano group of the compounds (Ia-If) under acidic or basic conditions. 
The hydrolysis reaction in this reaction may be carried out according to a conventional reaction for hydrolysis of a nitrile group; for example, acid hydrolysis using hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, polyphosphoric acid, etc. alone or in any optional combination therewith, and alkaline hydrolysis using lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, ammonia, etc.
The reaction may be carried out in the presence or absence of a solvent. The solvent which may be used includes methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, dioxane, tetrahydrofuran, diethyl ether, petroleum ether, n-hexane, cyclohexane, benzene, toluene, xylene, chlorobenzene, pyridine, ethyl acetate, N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform, carbon tetrachloride, water and the like.
The reagent and solvent to be used in the reaction, as well as use or no use of the solvent, should be properly selected, depending on the substrates and reaction parameters used.
The compound (Ig) may be prepared according to the following process:
The thiophene compounds (III) having an alkoxycarbonyl group instead of the cyano group may be prepared by using a halogenated acetic acid ester such as methyl bromoacetate or ethyl bromoacetate instead of the haloacetonitrile such as chloroacetonitrile or bromoaceto -nitrile, which was used, in the above 1)-(1), for thioetherification of one of the sulfur atoms derived from carbon disulfide in the preparation step of the intermediate thiophene compounds (III) for the compounds (Ia-If).
Then, the alkoxycarbonyl group of the thiophene compound (III) is hydrolyzed to a carboxyl group under acidic or basic conditions, followed by condensed cyclization reaction using hydroxylamine to prepare the compounds (Ia-If) having a carboxyl group instead of the cyano group as in the above 1).
The above-mentioned hydrolysis may be carried out by a conventional reaction for hydrolyzing an ester; for example, acid hydrolysis using hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, polyphosphoric acid, etc. alone or in any optional combination therewith, and alkaline hydrolysis using lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, ammonia, etc.
This reaction may be carried out in the presence or absence of a solvent. The solvent which may be used includes methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, dioxane, tetrahydrofuran, diethyl ether, petroleum ether, n-hexane, cyclohexane, benzene, toluene, xylene, chlorobenzene, pyridine, ethyl acetate, N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform, carbon tetrachloride, water and the like.
The reagent and solvent to be used in the reaction, as well as use or no use of the solvent should be properly selected, depending on the substrates and reaction parameters used.
Amidation of the carboxyl group with an amino compound represented by HNR5R6 (wherein R5 and R6 have the same meanings as defined above) may finally give the compound (Ig) of the invention.
Amidation includes transesterification with an amine, a condensation reaction of a carboxylic acid derived from hydrolysis of an ester with an amine or the like. A condensing agent includes, for example, acid halides such as thionyl chloride, alkyl chlorocarbonates such as ethyl chlorocarbonate, carbodiimide compounds such as dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylamino)propylcarbodiimide, sulfonyl chloride compounds such as methanesulfonyl chloride, phosphorus compounds such as diphenyl phosphite, diphenylphosphoryl chloride, triphenylphosphine-diethyl azadicarboxylate, N,Nxe2x80x2-carbodiimidazole.
This reaction may be carried out in the presence or absence of a solvent. The solvent which may be used includes methanol, ethanol, n-propanol, isopropanol, n -butanol, t-butanol, dioxane, tetrahydrofuran, diethyl ether, petroleum ether, n-hexane, cyclohexane, benzene, toluene, xylene, chlorobenzene, pyridine, ethyl acetate, N,N -dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform, carbon tetrachloride, water and the like.
The reagent and solvent to be used in the reaction, as well as use or no use of the solvent should be properly selected, depending on the substrates and reaction parameters used.
3)The compounds (I) of the invention wherein n is 1 or 2 may be prepared by using a conventional oxidation reaction to oxidize the sulfur atom in the alkylthio group (xe2x80x94Sxe2x80x94R 2) of the compounds (Ia-Ig) to sulfoxide or sulfone. The oxidizing agent which may be used for the oxidation reaction includes, for example, hydrogen peroxide, t-butyl hydroperoxide, meta-chloroperbenzoic acid, peracetic acid, sodium meta-periodate, bromous acid sodium salt, sodium hypochlorite, periodobenzene, and the like.
This reaction may be carried out in the presence or absence of a solvent. The solvent which may be used includes methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, dioxane, tetrahydrofuran, diethyl ether, petroleum ether, n-hexane, cyclohexane, benzene, toluene, xylene, chlorobenzene, pyridine, ethyl acetate, N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform, carbon tetrachloride, water and the like.
The reagent and solvent to be used in the reaction, as well as use or no use of the solvent should be properly selected, depending on the substrates and reaction parameters used.
The compound of the present invention has a potent enhancing activity of osteogenesis and can be used as an enhancing agent for osteogenesis in repairing or transplanting the bone or alveolar bone, alone or in admixture with a carrier for bone repairing.
The compound of the invention, when used as an enhancing agent for osteogenesis, may be administered orally or parenterally in a dosage form of tablets, powders, solutions, injections, suppositories or others. It may also be directly applied to the bone that has been surgically removed. Optimum dose may be chosen by totally considering the age, sexuality, body weight and others of patients.
The compound of the invention, when used in admixture with a carrier for bone repairing, may be adhered onto or included in an artificial bone made of metals, ceramics or polymers. The artificial bone is preferably made to have porous surface so as to release the enhancing agent of osteoblast differentiation according to the invention in living tissues when the bone is transplanted to the defective part of bone.